NL3 TIE ligand homologue nucleic acids

ABSTRACT

The present invention concerns isolated nucleic acid molecules encoding the novel TIE ligands NL2, NL3 and FLS139, the proteins encoded by such nucleic acid molecules, as well as methods and means for making and using such nucleic acid and protein molecules.

This is a divisional of application Ser. No. 09/143,707, filed Aug. 28,1998, now U.S. Pat. No. 6,348,350, which claims benefit of U.S.Provisional Application No. 60/059,352, filed Sep. 19, 1997.

FIELD OF THE INVENTION

The present invention concerns isolated nucleic acid molecules encodingnovel TIE ligands, the TIE proteins encoded by such nucleic acidmolecules, as well as methods and means for making and using suchnucleic acid and protein molecules.

BACKGROUND ART

The abbreviations “TIE” or “tie” are acronyms, which stand for “tyrosinekinase containing Ig and EGF homology domains” and were coined todesignate a new family of receptor tyrosine kinases which are almostexclusively expressed in vascular endothelial cells and earlyhemopoietic cells, and are characterized by the presence of an EGF-likedomain, and extracellular folding units stabilized by intra-chaindisulfide bonds, generally referred to as “immunoglobulin (IG)-like”folds. A tyrosine kinase homologous cDNA fragment from human leukemiacells (tie) was described by Partanen et al., Proc. Natl. Acad. Sci. USA87, 8913-8917 (1990). The mRNA of this human “tie” receptor has beendetected in all human fetal and mouse embryonic tissues, and has beenreported to be localized in the cardiac and vascular endothelial cells.Korhonen et al., Blood 80, 2548-2555 (1992); PCT Application PublicationNo. WO 93/14124 (published Jul. 22, 1993). The rat homolog of human tie,referred to as “tie-1”, was identified by Maisonpierre et al., Oncogene8 1631-1637 (1993)). Another tie receptor, designated “tie-2” wasoriginally identified in rats (Dumont et al., Oncogene 8, 1293-1301(1993)), while the human homolog of tie-2, referred to as “ork” wasdescribed in U.S. Pat. No. 5,447,860 (Ziegler). The murine homolog oftie-2 was originally termed “tek.” The cloning of a mouse tie-2 receptorfrom a brain capillary cDNA library is disclosed in PCT ApplicationPublication No. WO 95/13387 (published May 28, 1995). The TIE receptorsare believed to be actively involved in angiogenesis, and may play arole in hemopoiesis as well.

The expression cloning of human TIE-2 ligands has been described in PCTApplication Publication No. WO 96/11269 (published Apr. 18, 1996) and inU.S. Pat. No. 5,521,073 (published May 28, 1996). A vector designated asλgt10 encoding a TIE-2 ligand named “htie-2 ligand 1” or “hTL1” has beendeposited under ATCC Accession No. 75928. A plasmid encoding anotherTIE-2 ligand designated “htie-2 2” or “hTL2” is available under ATCCAccession No. 75928. This second ligand has been described as anantagonist of the TIE-2 receptor. The identification of secreted humanand mouse ligands for the TIE-2 receptor has been reported by Davis etal., Cell 87 1161-1169 (1996). The human ligand designated“Angiopoietin-1”, to reflect its role in angiogenesis and potentialaction during hemopoiesis, is the same ligand as the ligand variouslydesignated as “htie-2 1” or “hTL-1” in WO 96/11269. Angiopoietin-1 hasbeen described to play an angiogenic role later and distinct from thatof VEGF (Suri et al., Cell 87, 1171-1180 (1996)). Since TIE-2 isapparently upregulated during the pathologic angiogenesis requisite fortumor growth (Kaipainen et al., Cancer Res. 54, 6571-6577 (1994))angiopoietin-1 has been suggested to be additionally useful forspecifically targeting tumor vasculature (Davis et al., supra).

SUMMARY OF THE INVENTION

The present invention concerns novel human TIE ligands with powerfuleffects on vasculature. The invention also provides for isolated nucleicacid molecules encoding such ligands or functional derivatives thereof,and vectors containing such nucleic acid molecules. The inventionfurther concerns host cells transformed with such nucleic acid toproduce the novel TIE ligands or functional derivatives thereof. Thenovel ligands may be agonists or antagonists of TIE receptors, known orhereinafter discovered. Their therapeutic or diagnostic use, includingthe delivery of other therapeutic or diagnostic agents to cellsexpressing the respective TIE receptors, is also within the scope of thepresent invention.

The present invention further provides for agonist or antagonistantibodies specifically binding the TEE ligands herein, and thediagnostic or therapeutic use of such antibodies.

In another aspect, the invention concerns compositions comprising thenovel ligands or antibodies.

In a further aspect, the invention concerns conjugates of the novel TIEligands of the present invention with other therapeutic or cytotoxicagents, and compositions comprising such conjugates. Because the TIE-2receptor has been reported to be upregulated during the pathologicangiogenesis that is requisite for tumor growth, the conjugates of theTIE ligands of the present invention to cytotoxic or other anti-tumoragents are useful in specifically targeting tumor vasculature.

In yet another aspect, the invention concerns a method for identifying acell that expresses a TIE (e.g. TIE-2) receptor, which comprisescontacting a cell with a detectably labeled TIE ligand of the presentinvention under conditions permitting the binding of such TIE ligand tothe TIE receptor, and determining whether such binding has indeedoccurred.

In a different aspect, the invention concerns a method for measuring theamount of a TIE ligand of the present invention in a biological sampleby contacting the biological sample with at least one antibodyspecifically binding the TIE ligand, and measuring the amount of the TIEligand-antibody complex formed.

The invention further concerns a screening method for identifyingpolypeptide or small molecule agonists or antagonists of a TIE receptorbased upon their ability to compete with a native or variant TIE ligandof the present invention for binding to a corresponding TIE receptor.

The invention also concerns a method for imaging the presence ofangiogenesis in wound healing, in inflammation or in tumors of humanpatients, which comprises administering detectably labeled TIE ligandsor agonist antibodies of the present invention, and detectingangiogenesis.

In another aspect, the invention concerns a method of promoting orinhibiting neovascularization in a patient by administering an effectiveamount of a TIE ligand of the present invention in a pharmaceuticallyacceptable vehicle. In a preferred embodiment, the present inventionconcerns a method for the promotion of wound healing. In anotherembodiment, the invention concerns a method for promoting angiogenicprocesses, such as for inducing collateral vascularization in anischemic heart or limb. In a further preferred embodiment, the inventionconcerns a method for inhibiting tumor growth.

In yet another aspect, the invention concerns a method of promoting bonedevelopment and/or maturation and/or growth in a patient, comprisingadministering to the patient an effective amount of a TIE ligand of thepresent invention in a pharmaceutically acceptable vehicle.

In a further aspect, the invention concerns a method of promoting musclegrowth and development, which comprises administering a patient in needan effective amount of a TIE ligand of the present invention in apharmaceutically acceptable vehicle.

The TIE ligands of the present invention may be administered alone, orin combination with each other and/or with other therapeutic ordiagnostic agents, including members of the VEGF family. Combinationstherapies may lead to new approaches for promoting or inhibitingneovascularization, and muscle growth and development.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic depiction of the relationship of the ligands NL2,NL3 and FLS 139 with the two known ligands of the TIE2 receptor(h-TEE2L1 and h-TIE2L2) and with other TIE ligands disclosed inapplication Ser. No. 08/933,821 filed at equal date (attorney docket no:1130).

FIGS. 2A and 2B are the nucleotide sequence of the TIE ligand NL2 (SEQ.ID. NO: 1) (DNA 22780).

FIGS. 3A and 3B are the amino acid sequence of the TIE ligand NL2 (SEQ.ID. NO:2).

FIG. 4 is the nucleotide sequence of the TIE ligand NL3 (SEQ. ID. NO: 3)(DNA 33457).

FIG. 5 is the amino acid sequence of the TIE ligand NL3 (SEQ. ID. NO:4).

FIGS. 6A and 6B are the nucleotide sequence of the TIE ligand FLS 139(SEQ. ID NO: 5) (DNA 16451).

FIGS. 7A and 7B are the amino acid sequence of the TIE ligand FLS 139(SEQ. ID NO:6).

FIGS. 8-9 -Northern blots showing the expression of the mRNAs of TIEligands NL2 and NL3 in various tissues.

DETAILED DESCRIPTION OF THE INVENTION A. TIE LIGANDS AND NUCLEIC ACIDMOLECULES ENCODING THEM

The TIE ligands of the present invention include the native humanligands designated NL2 (SEQ. ID. NO: 2), NL3 (SEQ. ID. NO: 4), and FLS139 (SEQ. ID. NO: 6), their homologs in other, non-human mammalianspecies, including, but not limited to, higher mammals, such as monkey;rodents, such as mice, rats, hamster; porcine; equine; bovine; naturallyoccurring allelic and splice variants, and biologically active(functional) derivatives, such as, amino acid sequence variants of suchnative molecules, as long as they differ from a native TL-1 or TL-2ligand. Native NL2, as disclosed herein, has 27% amino acid sequenceidentity with hTL-1 (TIE2L1) and about 24% amino acid sequence identitywith hTL-2 (TIE2L2). The amino acid sequence of native NL3, as disclosedherein, is about 30% identical with that of hTL-1 and about 29%identical with that of hTL-2. The amino acid sequence identity betweennative FLS 139, as disclosed herein, and hTL-1 and h-TL2 is about 21%.The native TIE ligands of the present invention are substantially freeof other proteins with which they are associated in their nativeenvironment. This definition is not limited in any way by the method(s)by which the TIE ligands of the present invention are obtained, andincludes all ligands otherwise within the definition, whether purifiedfrom natural source, obtained by recombinant DNA technology,synthesized, or prepared by any combination of these and/or othertechniques. The amino acid sequence variants of the native TIE ligandsof the present invention shall have at least about 90%, preferably, atleast about 95%, more preferably at least about 98%, most preferably atleast about 99% sequence identity with a full-length, native human TIEligand of the present invention, or with the fibrinogen-like domain of anative human TIE ligand of the present invention. Such amino acidsequence variants preferably exhibit or inhibit a qualitative biologicalactivity of a native TIE ligand.

The term “fibrinogen domain” or “fibrinogen-like domain” is used torefer to amino acids from about position 278 to about position 498 inthe known hTL-1 amino acid sequence; amino acids from about position 276to about position 496 in the known hTL-2 amino acid sequence; aminoacids from about position 180 to about 406 in the amino acid sequence ofNL2; amino acids from about position 77 to about position 288 in theamino acid sequence of NL3; and amino acids from about position 238 toabout position 460 in the amino acid sequence of FLS139, and tohomologous domains in other TIE ligands. The fibrinogen-like domain ofNL2 is about 37-38% identical to that of the hTL-1 (TIE2L1) and hTL-2(TEE2L2). The NL3 fibrinogen-like domain is about 37% identical to thefibrinogen-like domains of hTL-1 and hTL-2, while the FLS 139fibrinogen-like domain is about 32-33% identical to the fibrinogen-likedomains of hTL-1 and hTL-2.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given TIE ligandmay be produced. The present invention specifically contemplates everypossible variation of nucleotide sequences, encoding the TIE ligands ofthe present invention, based upon all possible codon choices. Althoughnucleic acid molecules which encode the TIE ligands herein arepreferably capable of hybridizing, under stringent conditions, to anaturally occurring TIE ligand gene, it may be advantageous to producenucleotide sequences encoding TIE ligands, which possess a substantiallydifferent codon usage. For example, codons may be selected to increasethe rate at which expression of the polypeptide occurs in a particularprokaryotic or eukaryotic host cells, in accordance with the frequencywith which a particular codon is utilized by the host. In addition, RNAtranscripts with improved properties, e.g. half-life can be produced byproper choice of the nucleotide sequences encoding a given TIE ligand.

“Sequence identity” shall be determined by aligning the two sequences tobe compared following the Clustal method of multiple sequence alignment(Higgins et al., Comput. Appl. Biosci. 5, 151-153 (1989), and Higgins etal., Gene 73, 237-244 (1988)) that is incorporated in version 1.6 of theLasergene biocomputing software (DNASTAR, Inc., Madison, Wis.), or anyupdated version or equivalent of this software.

The terms “biological activity” and “biologically active” with regard toa TIE ligand of the present invention refer to the ability of a moleculeto specifically bind to and signal through a native TIE receptor, e. g.a native TIE-2 receptor, or to block the ability of a native TIEreceptor (e.g. TIE-2) to participate in signal transduction. Thus, the(native and variant) TIE ligands of the present invention includeagonists and antagonists of a native TIE, e.g. TIE-2, receptor.Preferred biological activities of the TIE ligands of the presentinvention include the ability to induce or inhibit vascularization. Theability to induce vascularization will be useful for the treatment ofbiological conditions and diseases, where vascularization is desirable,such as wound healing, ischaemia, and diabetes. On the other hand, theability to inhibit or block vascularization may, for example, be usefulin preventing or attenuating tumor growth. Another preferred biologicalactivity is the ability to affect muscle growth or development. Afurther preferred biological activity is the ability to influence bonedevelopment, maturation, or growth.

The term “functional derivative” is used to define biologically activeamino acid sequence variants of the native TIE ligands of the presentinvention, as well as covalent modifications, including derivativesobtained by reaction with organic derivatizing agents,post-translational modifications, derivatives with nonproteinaceouspolymers, and immunoadhesins.

The term “isolated” when used to describe the various polypeptidesdescribed herein, means polypeptides that have been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the TIE ligand'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes nucleic acid molecules contained in cells that ordinarilyexpress an TIE ligand of the present invention, where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.Deletional variants include those having C- and/or N-terminal deletions(truncations) as well as variants with internal deletions of one or moreamino acids. The preferred deletional variants of the present inventioncontain deletions outside the fibrinogen-like domain of a native TIEligand of the present invention.

The amino acid sequence variants of the present invention may containvarious combinations of amino acid substitutions, insertions and/ordeletions, to produce molecules with optimal characteristics.

The amino acids may be classified according to the chemical compositionand properties of their side chains. They are broadly classified intotwo groups, charged and uncharged. Each of these groups is divided intosubgroups to classify the amino acids more accurately.

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions areexpected to result in significant changes in the biologicalproperties/function of the obtained variant Amino acid sequencedeletions generally range from about 1 to 30 residues, more preferablyabout 1 to 10 residues, and typically are contiguous. Deletions may beintroduced into regions not directly involved in the interaction with anative TIE receptor. Deletions are preferably performed outside thefibrinogen-like regions at the C-terminus of the TIE ligands of thepresent invention.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the TIE ligand amino acid sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5 residues, more preferably1 to 3 residues. Examples of terminal insertions include the TIE ligandswith an N-terminal methionyl residue, an artifact of its directexpression in bacterial recombinant cell culture, and fusion of aheterologous N-terminal signal sequence to the N-terminus of the TIEligand molecule to facilitate the secretion of the mature TIE ligandfrom recombinant host cells. Such signal sequences will generally beobtained from, and thus homologous to, the intended host cell species.Suitable sequences include, for example, STII or Ipp for E. coli, alphafactor for yeast, and viral signals such as herpes gD for mammaliancells. Other insertional variants of the native TIE ligand moleculesinclude the fusion of the N- or C-terminus of the TIE ligand molecule toimmunogenic polypeptides, e.g. bacterial polypeptides such asbeta-lactamase or an enzyme encoded by the E. coli trp locus, or yeastprotein, and C-terminal fusions with proteins having a long half-lifesuch as immunoglobulin regions (preferably immunoglobulin constantregions), albumin, or ferritin, as described in WO 89/02922 published onApr. 6, 1989.

Since it is often difficult to predict in advance the characteristics ofa variant TIE ligand, it will be appreciated that some screening will beneeded to select the optimum variant.

Amino acid sequence variants of native TIE ligands of the presentinvention are prepared by methods known in the art by introducingappropriate nucleotide changes into a native or variant TIE ligand DNA,or by in vitro synthesis of the desired polypeptide. There are twoprincipal variables in the construction of amino acid sequence variants:the location of the mutation site and the nature of the mutation. Withthe exception of naturally-occurring alleles, which do not require themanipulation of the DNA sequence encoding the TIE ligand, the amino acidsequence variants of TIE are preferably constructed by mutating the DNA,either to arrive at an allele or an amino acid sequence variant thatdoes not occur in nature.

One group of the mutations will be created within the domain or domainsof the TIE ligands of the present invention identified as being involvedin the interaction with a TIE receptor, e.g. TIE-1 or TIE-2.

Alternatively or in addition, amino acid alterations can be made atsites that differ in TIE ligands from various species, or in highlyconserved regions, depending on the goal to be achieved.

Sites at such locations will typically be modified in series, e.g. by(1) substituting first with conservative choices and then with moreradical selections depending upon the results achieved, (2) deleting thetarget residue or residues, or (3) inserting residues of the same ordifferent class adjacent to the located site, or combinations of options1-3.

One helpful technique is called “alanine scanning” (Cunningham andWells, Science 244, 1081-1085 [1989]). Here, a residue or group oftarget residues is identified and substituted by alanine or polyalanine.Those domains demonstrating functional sensitivity to the alaninesubstitutions are then refined by introducing further or othersubstituents at or for the sites of alanine substitution.

After identifying the desired mutation(s), the gene encoding an aminoacid sequence variant of a TIE ligand can, for example, be obtained bychemical synthesis as hereinabove described.

More preferably, DNA encoding a TIE ligand amino acid sequence variantis prepared by site-directed mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the ligand. Site-directed(site-specific) mutagenesis allows the production of ligand variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechniques of site-specific mutagenesis are well known in the art, asexemplified by publications such as, Edelman et al., DNA 2, 183 (1983).As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al, Third Cleveland Symposium on Macromoleculesand Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). Thisand other phage vectors are commercially available and their use is wellknown to those skilled in the art. A versatile and efficient procedurefor the construction of oligodeoxyribonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller, M. J. and Smith, M., Nucleic Acids Res. 10, 6487-6500 [1982]).Also, plasmid vectors that contain a single-stranded phage origin ofreplication (Veira et al., Meth. Enzymol. 153, 3 [1987]) may be employedto obtain single-stranded DNA. Alternatively, nucleotide substitutionsare introduced by synthesizing the appropriate DNA fragment in vitro,and amplifying it by PCR procedures known in the art.

In general, site-specific mutagenesis herewith is performed by firstobtaining a single-stranded vector that includes within its sequence aDNA sequence that encodes the relevant protein. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., Proc. Natl.Acad. Sci. USA 75, 5765 (1978). This primer is then annealed with thesingle-stranded protein sequence-containing vector, and subjected toDNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment,to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells such as JP101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region may be removed and placed in anappropriate expression vector for protein production.

The PCR technique may also be used in creating amino acid sequencevariants of a TIE ligand. When small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more) partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp^(R) kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayered with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Tag) DNA polymerase (5 units/ 1), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows:

2 min. 55° C.,

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.,

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. [Gene 34, 315 (1985)]. Thestarting material is the plasmid (or vector) comprising the TIE ligandDNA to be mutated. The codon(s) within the TIE ligand to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the DNA encoding the TIE ligand. After therestriction sites have been introduced into the plasmid, the plasmid iscut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction site butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated TIE ligand DNA sequence.

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant TIE ligands.This method involves (a) constructing a replicable expression vectorcomprising a first gene encoding an receptor to be mutated, a secondgene encoding at least a portion of a natural or wild-type phage coatprotein wherein the first and second genes are heterologous, and atranscription regulatory element operably linked to the first and secondgenes, thereby forming a gene fusion encoding a fusion protein; (b)mutating the vector at one or more selected positions within the firstgene thereby forming a family of related plasmids; (c) transformingsuitable host cells with the plasmids; (d) infecting the transformedhost cells with a helper phage having a gene encoding the phage coatprotein; (e) culturing the transformed infected host cells underconditions suitable for forming recombinant phagemid particlescontaining at least a portion of the plasmid and capable of transformingthe host, the conditions adjusted so that no more than a minor amount ofphagemid particles display more than one copy of the fusion protein onthe surface of the particle; (f) contacting the phagemid particles witha suitable antigen so that at least a portion of the phagemid particlesbind to the antigen; and (g) separating the phagemid particles that bindfrom those that do not. Steps (d) through (g) can be repeated one ormore times. Preferably in this method the plasmid is under tight controlof the transcription regulatory element, and the culturing conditionsare adjusted so that the amount or number of phagemid particlesdisplaying more than one copy of the fusion protein on the surface ofthe particle is less than about 1%. Also, preferably, the amount ofphagemid particles displaying more than one copy of the fusion proteinis less than 10% of the amount of phagemid particles displaying a singlecopy of the fusion protein. Most preferably, the amount is less than20%. Typically in this method, the expression vector will furthercontain a secretory signal sequence fused to the DNA encoding eachsubunit of the polypeptide and the transcription regulatory element willbe a promoter system. Preferred promoter systems are selected from lacZ, λ_(PL), tac, T7 polymerase, tryptophan, and alkaline phosphatasepromoters and combinations thereof. Also, normally the method willemploy a helper phage selected from M13K07, M13R408, M13-VCS, and Phi X174. The preferred helper phage is M13K07, and the preferred coatprotein is the M13 Phage gene III coat protein. The preferred host is E.coli, and protease-deficient strains of E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,Molecular Cloning: A laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and Current Protocols in Molecular Biology,Ausubel et al., eds., Wiley-Interscience, 1991.

“Immunoadhesins” are chimeras which are traditionally constructed from areceptor sequence linked to an appropriate immunoglobulin constantdomain sequence (immunoadhesins). Such structures are well known in theart. Immunoadhesins reported in the literature include fusions of the Tcell receptor* [Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,2936-940 (1987)]; CD4* [Capon et al, Nature 337,525-531 (1989);Traunecker et al, Nature 339, 68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 92 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990)];L-selectin (homing receptor) [Watson et al, J. Cell. Biol. 110,2221-2229 (1990); Watson et al., Nature 349, 164-167 (1991)]; CD44*[Aruffo et al, Cell 61, 1303-1313 (1990)]; CD28* and B7* [Linsley et al.J. Exp. Med. 173, 721-730 (1991)]; CTLA-4* [Lisley et al., J. Exp. Med.174, 561-569 (1991)]; CD22* [Stamenkovic et al., Cell 66. 1133-1144(1991)]; TNF receptor [Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88,10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27, 2883-2886(1991); Peppel et al, J. Exp. Med. 174, 1483-1489 (1991)]; NP receptors[Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptorα-chain* [Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)];HGF receptor [Mark, M. R. et al., 1992, J. Biol. Chem. submitted], wherethe asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

Ligand-immunoglobulin chimeras are also known, and are disclosed, forexample, in U.S. Pat. No. 5,304,640 (for L-selectin ligands); U.S. Pat.Nos. 5,316,921 and 5,328,837 (for HGF variants). These chimeras can bemade in a similar way to the construction of receptor-immunoglobulinchimeras.

Covalent modifications of the TIE ligands of the present invention areincluded within the scope herein. Such modifications are traditionallyintroduced by reacting targeted amino acid residues of the TIE ligandwith an organic derivatizing agent that is capable of reacting withselected sides or terminal residues, or by harnessing mechanisms ofpost-translational modifications that function in selected recombinanthost cells. The resultant covalent derivatives are useful in programsdirected at identifying residues important for biological activity, forimmunoassays, or for the preparation of anti-TIE ligand antibodies forimmunoaffinity purification of the recombinant. For example, completeinactivation of the biological activity of the protein after reactionwith ninhydrin would suggest that at least one arginyl or lysyl residueis critical for its activity, whereafter the individual residues whichwere modified under the conditions selected are identified by isolationof a peptide fragment containing the modified amino acid residue. Suchmodifications are within the ordinary skill in the art and are performedwithout undue experimentation.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl4-ethyl) carbodiimide or1-ethyl-3-(4-azonia4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with anmmonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86[1983]), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the TIE ligand with polypeptides as well asfor cross-linking the TIE ligand polypeptide to a water insolublesupport matrix or surface for use in assays or affinity purification. Inaddition, a study of interchain cross-links will provide directinformation on conformational structure. Commonly used cross-linkingagents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio] propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the a -amino groups of lysine,arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Francisco,pp. 79-86 (1983)].

Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

The TIE ligands may be linked to various nonproteinaceous polymers, suchas polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes,in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. These variants, just asthe immunoadhesins of the present invention are expected to have longerhalf-lives than the corresponding native TIE ligands.

The TIE ligands may be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

The term “native TIE receptor” is used herein to refer to a TIE receptorof any animal species, including, but not limited to, humans, otherhigher primates, e.g. monkeys, and rodents, e.g. rats and mice. Thedefinition specifically includes the TIE-2 receptor, disclosed, forexample, in PCT Application Serial No. WO 95/13387 (published May 18,1995), and the endothelial cell receptor tyrosine kinase termed “TIE” inPCT Application Publication No. WO 93/14124 (published Jul. 22 1993),and preferably is TIE-2.

B. ANTI-TIE LIGAND ANTIBODIES

The present invention covers agonist and antagonist antibodies,specifically binding the TIE ligands. The antibodies may be monoclonalor polyclonal, and include, without limitation, mature antibodies,antibody fragments (e.g. Fab, F(ab′)₂, F_(v), etc.), single-chainantibodies and various chain combinations.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist, antagonist, andneutralizing antibodies) specifically binding a TIE ligand of thepresent invention and antibody compositions with polyepitopicspecificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-TIE ligand antibody with a constant domain (e.g.“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv), so long as they exhibit the desired biologicalactivity. See, e.g. U.S. Pat. No. 4,816,567 and Mage et al., inMonoclonal Antibody Production Techniques and Applications, pp.79-97(Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

Polyclonal antibodies to a TIE ligand of the present invention generallyare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of the TIE ligand and an adjuvant.

It may be useful to conjugate the TIE ligand or a fragment containingthe target amino acid sequence to a protein that is immunogenic in thespecies to be immunized, e.g. keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glytaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-TIE ligand antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal boosted with the conjugate ofthe same TIE ligand, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-TIE ligand monoclonal antibodies of the inventionmay be made using the hybridoma method first described by Kohler &Milstein, Nature 256:495 (1975), or may be made by recombinant DNAmethods [Cabilly, et al., U.S. Pat. No. 4,816,567].

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell [Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Maryland USA. Human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies [Kozbor, J. Immunol. 133:3001 (1984); Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)].

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the TIE ligand.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard, Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of an anti-TIE ligand monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a TIEligand of the present invention and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercapatobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a TIE ligand or an immunologically reactive portionthereof) to compete with the test sample analyte (TIE ligand) forbinding with a limited amount of antibody. The amount of TIE ligand inthe test sample is inversely proportional to the amount of standard thatbecomes bound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al, Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen eta., Science 239, 1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/15,272 filedJun. 14, 1991.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immnunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255(1993); Jakobovits et al., Nature 362, 255-258 (1993).

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aparticular TIE ligand, the other one is for any other antigen, andpreferably for another ligand. For example, bispecific antibodiesspecifically binding two different TIE ligands are within the scope ofthe present invention.

Methods for making bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al, EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, and second and thirdconstant regions of an immunoglobulin heavy chain (CH2 and CH3). It ispreferred to have the first heavy chain constant region (CH1) containingthe site necessary for light chain binding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are cotransfected into a suitable host organism.This provides for great flexibility in adjusting the mutual proportionsof the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in copendingapplication Ser. No. 07/931,811 filed Aug. 17, 1992.

For further details of generating bispecific antibodies see, forexample, Suresh et al, Methods in Enzymology 121, 210 (1986).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

The term “agonist” is used to refer to peptide and non-peptide analogsof the native TIE ligands of the present invention and to antibodiesspecifically binding such native TIE ligands, provided that they havethe ability to signal through a native TIE receptor (e.g. TIE-2). Inother words, the term “agonist” is defined in the context of thebiological role of the TIE receptor, and not in relation to thebiological role of a native TIE ligand, which, as noted before, may bean agonist or antagonist of the TIE receptor biological function.Preferred agonists are promoters of vascularization.

The term “antagonist” is used to refer to peptide and non-peptideanalogs of the native TIE ligands of the present invention and toantibodies specifically binding such native TIE ligands, provided thatthey have the ability to inhibit the biological function of a native TIEreceptor (e.g. TIE-2). Again, the term “antagonist” is defined in thecontext of the biological role of the TIE receptor, and not in relationto the biological activity of a native TIE ligand, which may be eitheran agonist or an antagonist of the TIE receptor biological function.Preferred antagonists are inhibitors of vasculogenesis.

C. CLONING AND EXPRESSION OF THE TIE LIGANDS

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of the TIE ligand molecule that is inserted into thevector. If the signal sequence is heterologous, it should be selectedsuch that it is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell.

Heterologous signal sequences suitable for prokaryotic host cells arepreferably prokaryotic signal sequences, such as the α-amylase, ompA,ompC, ompE, ompF, alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the yeastinvertase, amylase, alpha factor, or acid phosphatase leaders may, forexample, be used. In mammalian cell expression mammalian signalsequences are most suitable. The listed signal sequences are forillustration only, and do not limit the scope of the present inventionin any way.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenabled the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequence are well known for a variety of bacteria, yeast and viruses.The origin of replication from the well-known plasmid pBR322 is suitablefor most gram negative bacteria, the 2μ plasmid origin for yeast andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins of replication are notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter). Most expressionvectors are “shuttle” vectors, i.e. they are capable of replication inat least one class of organisms but can be transfected into anotherorganism for expression. For example, a vector is cloned in E. coli andthen the same vector is transfected into yeast or mammalian cells forexpression even though it is not capable of replicating independently ofthe host cell chromosome.

DNA is also cloned by insertion into the host genome. This is readilyaccomplished using Bacillus species as hosts, for example, by includingin the vector a DNA sequence that is complementary to a sequence foundin Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theDNA encoding the desired heterologous polypeptide. However, the recoveryof genomic DNA is more complex than that of an exogenously replicatedvector because restriction enzyme digestion is required to excise theencoded polypeptide molecule.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forbacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin [Southern et al., J. Molec. Appl. Genet. 1, 327(1982)], mycophenolic acid [Mulligan et al, Science 209, 1422 (1980)],or hygromycin [Sudgen et al, Mol. Cel. Biol. 5,410-413 (1985)]. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Other examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the desirednucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the desired polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the desiredpolypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumwhich lacks hypoxanthine, glycine, and thymidine. An appropriate hostcell in this case is the Chinese hamster ovary (CHO) cell line deficientin DHFR activity, prepared and propagated as described by Urlaub andChasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). A particularlyuseful DHFR is a mutant DHFR that is highly resistant to MTX (EP117,060). This selection agent can be used with any otherwise suitablehost, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR. The DNA encoding DHFR and the desired polypeptide,respectively, then is amplified by exposure to an agent (methotrexate,or MTX) that inactivates the DHFR. One ensures that the cell requiresmore DHFR (and consequently amplifies all exogenous DNA) by selectingonly for cells that can grow in successive rounds of ever-greater MTXconcentration. Alternatively, hosts co-transformed with genes encodingthe desired polypeptide, wild-type DHFR, and another selectable markersuch as the neo gene can be identified using a selection agent for theselectable marker such as G418 and then selected and amplified usingmethotrexate in a wild-type host that contains endogenous DHFR. (Seealso U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al, 1979, Nature 282:39; Kingsmanet al., 1979, Gene 7:141; or Tschemper et al., 1980, Gene 10:157). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors, unlike cloning vectors, should contain a promoterwhich is recognized by the host organism and is operably linked to thenucleic acid encoding the desired polypeptide. Promoters areuntranslated sequences located upstream from the start codon of astructural gene (generally within about 100 to 1000 bp) that control thetranscription and translation of nucleic acid under their control. Theytypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to DNA encoding the desired polypeptide by removing themfrom their gene of origin by restriction enzyme digestion, followed byinsertion 5′ to the start codon for the polypeptide to be expressed.This is not to say that the genomic promoter for a TIE ligand is notusable. However, heterologous promoters generally will result in greatertranscription and higher yields of expressed TIE ligands as compared tothe native TIE ligand promoters.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al, Nature 281:544 (1979)), alkaline phosphatase,a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res. 8:4057(1980) and EPO Appln. Publ. No. 36,776) and hybrid promoters such as thetac promoter (H. de Boer et al, Proc. Nat'l. Acad. Sci. USA 80:21-25(1983)). However, other known bacterial promoters are suitable. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to DNA encoding a TIE ligand (Siebenlistet al, Cell 20:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding a TIE ligand.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1978); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

TIE ligand transcription from vectors in mammalian host cells may becontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat shock promoters, and from the promoter normally associated with theTIE ligand sequence, provided such promoters are compatible with thehost cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment which also contains the SV40 viralorigin of replication [Fiers et al., Nature 273:113 (1978), Mulligan andBerg, Science 209, 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA 78, 7398-7402 (1981)]. The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment [Greenaway et al., Gene 18, 355-360 (1982)]. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso, Gray et al., Nature 295, 503-508 (1982) on expressing cDNAencoding human immune interferon in monkey cells; Reyes et al., Nature297, 598-601 (1982) on expressing human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79, 5166-5170 (1982)on expression of the human interferon β1 gene in cultured mouse andrabbit cells; and Gorman et al., Proc. Natl. Acad. Sci., USA 79,6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse HIN-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the TIE ligands of the present inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent having been found 5′ [Laimins et al, Proc. Natl. Acad. Sci.USA 78,993 (1981)] and 3′ [Lasky et al, Mol Cel. Biol. 3, 1108 (1983)]to the transcription unit, within an intron [Banerji et al, Cell 33,729(1983)] as well as within the coding sequence itself [Osborne et al,Mol. Cel. Biol. 4, 1293 (1984)]. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297, 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theTIE ligand DNA, but is preferably located at a site 5′ from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the TIE ligand. The 3′ untranslated regionsalso include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components, the desired coding and control sequences, employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TIE ligand. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient systems,comprising a suitable expression vector and a host cell, allow for theconvenient positive identification of polypeptides encoded by clonesDNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of a TIE ligand.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the TIE polypeptides in recombinant vertebrate cell cultureare described in Getting et al., Nature 293, 620-625 (1981); Mantel etal., Nature 281, 4046 (1979); Levinson et al; EP 117,060 and EP 117,058.A particularly useful plasmid for mammalian cell culture expression ofthe TIE ligand polypeptides is pRK5 (EP 307,247), along with itsderivatives, such as, pRK5D that has an sp6 transcription initiationsite followed by an SfiI restriction enzyme site preceding the Xho/NotlIcDNA cloning sites, and pRK5B, a precursor of pRK5D that does notcontain the SfiI site; see, Holmes et al., Science 253, 1278-1280(1991).

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequences by the methods of Messing et al, NucleiAcids Res. 9, 309 (1981) or by the method of Maxam et al, Methods inEnzymology 65,499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TIE ligand. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high level of a desiredpolypeptide encoded by the expression vector. Sambrook et al, supra, pp.16.17-16.22. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positivescreening of such polypeptides for desired biological or physiologicalproperties. Thus transient expression systems are particularly useful inthe invention for purposes of identifying analogs and variants of nativeTIE ligands with the requisite biological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of a TIE ligand (including functional derivatives of nativeproteins) in recombinant vertebrate cell culture are described inGething et al., Nature 293 620-625 (1981); Mantei et al., Nature 281,40-46 (1979); Levinson et al, EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of a TIE ligand ispRK5 (EP 307,247) or pSVI6B (PCT Publication No. WO 91/08291).

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast or higher eukaryote cells described above. Suitableprokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B. E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia Marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe[Beach and Nurse, Nature 290, 140 (1981)], Kluyveromyces lactis[Louvencourt et al., J. Bacteriol. 737 (1983)]; yarrowia (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa [Case et al, Proc. Natl. Acad. Sci. USA 76, 5259-5263(1979)]; and Aspergillus hosts such as A. nidulans [Ballance et al,Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al, Gene26, 205-221 (1983); Yelton et al, Proc. Natl. Acad. Sci. USA 81,1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4, 475-479(1985)].

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegpti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6,47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al, eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa califomica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Generally, plant cells are transfected by incubation with certainstrains of the bacterium Agrobacterium tumefaciens, which has beenpreviously manipulated to contain the TIE ligand DNA. During incubationof the plant cell culture with A. tumefaciens, the DNA encoding a TIEligand is transferred to the plant cell host such that it istransfected, and will, under appropriate conditions, express the TIEligand DNA. In addition, regulatory and signal sequences compatible withplant cells are available, such as the nopaline synthase promoter andpolyadenylation signal sequences. Depicker et al, J. Mol. Appl. Gen. 1,561 (1982). In addition, DNA segments isolated from the upstream regionof the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. See EP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known.See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cellline [293 or 293 cells subcloned for growth in suspension culture,Graham et al, J. Gen. Virol. 36, 59 (1977)]; baby hamster kidney cells9BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR [CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA 77,4216 (1980)]; mouse sertolli cells[TM4, Mather, Biol. Reprod. 23, 243-251 (1980)]; monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells[Mather et al., Annals N.Y. Acad. Sci. 383,44068 (1982)]; MRC 5 cells;FS4 cells; and a human hepatoma cell line (Hep G2). Preferred host cellsare human embryonic kidney 293 and Chinese hamster ovary cells.

Particularly preferred host cells for the purpose of the presentinvention are vertebrate cells producing the TIE ligands of the presentinvention.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as is appropriate for inducingpromoters or selecting transformants containing amplified genes.

Prokaryotes cells used to produced the TIE ligands of this invention arecultured in suitable media as describe generally in Sambrook et al,supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such Ham's F10(Sigma), Minimal Essential Medium (MEM,Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM,Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham and Wallace, Meth. Enzymol. 58, 44 (1979);Barnes and Sato, Anal. Biochem. 102, 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or U.S. Pat. Re. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the TIE ligands of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the particular TIE ligand.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al, Am. J. Clin. Pharm.75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native TIE ligand polypeptide of the present invention, oragainst a synthetic peptide based on the DNA sequence provided herein asdescribed further hereinbelow.

The TIE ligand may be produced in host cells in the form of inclusionbodies or secreted into the periplasmic space or the culture medium, andis typically recovered from host cell lysates. The recombinant ligandsmay be purified by any technique allowing for the subsequent formationof a stable protein.

When the TIE ligand is expressed in a recombinant cell other than one ofhuman origin, it is completely free of proteins or polypeptides of humanorigin. However, it is necessary to purify the TIE ligand fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogenous as to the ligand. As a first step, theculture medium or lysate is centrifuged to remove particulate celldebris. The membrane and soluble protein fractions are then separated.The TIE ligand may then be purified from the soluble protein fraction.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; and protein A Sepharose columns to remove contaminantssuch as IgG.

Functional derivatives of the TIE ligands in which residues have beendeleted, inserted and/or substituted are recovered in the same fashionas the native ligands, taking into account of any substantial changes inproperties occasioned by the alteration. For example, fusion of the TIEligand with another protein or polypeptide, e.g. a bacterial or viralantigen, facilitates purification; an immunoaffinity column containingantibody to the antigen can be used to absorb the fusion. Immunoaffinitycolumns such as a rabbit polyclonal anti-TIE ligand column can beemployed to absorb TIE ligand variants by binding to at least oneremaining immune epitope. A protease inhibitor, such as phenyl methylsulfonyl fluoride (PMSF) also may be useful to inhibit proteolyticdegradation during purification, and antibiotics may be included toprevent the growth of adventitious contaminants. The TIE ligands of thepresent invention are conveniently purified by affinity chromatography,based upon their ability to bind to a TIE receptor, e.g. TIE-2.

One skilled in the art will appreciate that purification methodssuitable for native TIE ligands may require modification to account forchanges in the character of a native TIE ligand or its variants uponexpression in recombinant cell culture

D. USE OF THE TIE LIGANDS, NUCLEIC ACID MOLECULES AND ANTIBODIES

The TIE ligands of the present invention are useful in promoting thesurvival and/or growth and/or differentiation of TIE receptor (e.g.TIE-2 receptor) expressing cells in cell culture.

The TIE ligands may be additionally used to identify cells which expressnative TIE receptors, e.g. the TIE-2 receptor. To this end, a detectablylabeled ligand is contacted with a target cell under conditionpermitting its binding to the TIE receptor, and the binding ismonitored.

The TIE ligands herein may also be used to identify molecules exhibitinga biological activity of a TIE ligand, for example, by exposing a cellexpressing a TIE ligand herein to a test molecule, and detecting thespecific binding of the test molecule to a TIE (e.g. TIE-2) receptor,either by direct detection, or base upon secondary biological effects.This approach is particularly suitable for identifying new members ofthe TIE ligand family, or for screening peptide or non-peptide smallmolecule libraries.

The TIE ligands disclosed herein are also useful in screening assaysdesigned to identify agonists or antagonists of a native TIE (e.g.TIE-2) receptor, which promote or inhibit angiogenesis, and/or play animportant role in muscle growth or development and/or bone development,maturation or growth. For example, antagonists of the TIE-2 receptor maybe identified based upon their ability to block the binding of a TIEligand of the present invention to a native TIE receptor, as measured,for example, by using BiAcore biosensor technology (BIAcore; PharmaciaBiosensor, Midscataway, N.J.); or by monitoring their ability to blockthe biological response caused by a biologically active TIE ligandherein. Biological responses that may be monitored include, for example,the phosphorylation of the TIE-2 receptor or downstream components ofthe TIE-2 signal transduction pathway, or survival, growth ordifferentiation of cells expressing the TIE-2 receptor. Cell-basedassays, utilizing cells that do not normally the TIE-2 receptor,engineered to express this receptor, or to coexpress the TIE-2 receptorand a TIE ligand of the present invention, are particularly convenientto use.

In a particular embodiment, small molecule agonists and antagonists of anative TIE receptor, e.g. the TIE-2 receptor, can be identified, basedupon their ability to interfere with the TIE ligand/TIE receptorinteraction. There are numerous ways for measuring the specific bindingof a test molecule to a TIE receptor, including, but not limited todetecting or measuring the amount of a test molecule bound to thesurface of intact cells expressing the TIE receptor, cross-linked to theTIE receptor in cell lysates, or bound to the TIE receptor in vitro.

Detectably labeled TIE ligands include, for example, TIE ligandscovalently or non-covalently linked to a radioactive substances, e.g.¹²⁵I, a fluorescent substance, a substance having enzymatic activity(preferably suitable for colorimetric detection), a substrate for anenzyme (preferably suitable for colorimetric detection), or a substancethat can be recognized by a(n) (detectably labeled) antibody molecule.

The assays of the present invention may be performed in a manner similarto that described in PCT Publication WO 96/11269, published Apr. 18,1996.

The TIE ligands of the present invention are also useful for purifyingTIE receptors, e.g. TIE-2 receptors, optionally used in the form ofimmunoadhesins, in which the TIE ligand or the TIE receptor bindingportion thereof is fused to an immunoglobulin heavy or light chainconstant region.

The nucleic acid molecules of the present invention are useful fordetecting the expression of TIE ligands in cells or tissue sections.Cells or tissue sections may be contacted with a detectably labelednucleic acid molecule encoding a TIE ligand of the present inventionunder hybridizing conditions, and the presence of mRNA hybridized to thenucleic acid molecule determined, thereby detecting the expression ofthe TIE ligand.

Antibodies of the present invention may, for example, be used inimmunoassays to measure the amount of a TIE ligand in a biologicalsample. The biological sample is contacted with an antibody or antibodymixture specifically binding the a TIE ligand of the present invention,and the amount of the complex formed with a ligand present in the testsample is measured.

Antibodies to the TIE ligands herein may additionally be used for thedelivery of cytotoxic molecules, e.g. radioisotopes or toxins, ortherapeutic agents to cells expressing a corresponding TIE receptor. Thetherapeutic agents may, for example, be other TIE ligands, including theTIE-2 ligand, members of the vascular endothelial growth factor (VEGF)family, or known anti-tumor agents, and agents known to be associatedwith muscle growth or development, or bone development, maturation, orgrowth.

Anti-TIE ligand antibodies are also suitable as diagnostic agents, todetect disease states associated with the expression of a TIE (e.g.TIE-2) receptor. Thus, detectably labeled TIE ligands and antibodyagonists of a TIE receptor can be used for imaging the presence ofantiogenesis.

In addition, the new TIE ligands herein can be used to promoteneovascularization, and may be useful for inhibiting tumor growth.

Further potential therapeutic uses include the modulation of muscle andbone development, maturation, or growth.

For therapeutic use, the TIE ligands or anti-TIE ligand antibodies ofthe present invention are formulated as therapeutic compositioncomprising the active ingredient(s) in admixture with apharmacologically acceptable vehicle, suitable for systemic or topicalapplication. The pharmaceutical compositions of the present inventionare prepared for storage by mixing the active ingredient having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as Tween,Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983,“Biopolymers” 22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R.Langer, et al., 1981, “J. Biomed. Mater. Res.” 15: 167-277 and R.Langer, 1982, Chem. Tech.” 12: 98-105), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121A; Epstein et al., 1985,“Proc. Natl. Acad. Sci. USA” 82: 3688-3692; Hwang et al., 1980, “Proc.Natl. Acad. Sci. USA” 77: 4030-4034; EP 52322A; EP 36676A; EP 88046A; EP143949A; EP 142641A; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT-4 therapy.

An effective amount of a molecule of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

Further details of the invention will be apparent from the followingnon-limiting examples.

EXAMPLE 1

Identification of the FLS 139 Ligand

FLS 139 was identified in a cDNA library prepared from human fetal livermRNA obtained from Clontech Laboratories, Inc. Palo Alto, Calif. USA,catalog no. 64018-1, following the protocol described in “InstructionManual: Superscript® Lambda System for cDNA Synthesis and λ cloning,”cat. No. 19643-014, Life Technologies, Gaithersburg, Md., USA which isherein incorporated by reference. Unless otherwise noted, all reagentswere also obtained from Life Technologies. The overall procedure can besummarized into the following steps: (1) First strand synthesis; (2)Second strand synthesis; (3) Adaptor addition; (4) Enzymatic digestion;(5) Gel isolation of cDNA; (6) Ligation into vector; and (7)Transformation.

First Strand Synthesis:

Not1 primer-adapter (Life Tech., 2 μl, 0.5 μg) was added to a sterile1.5 ml microcentrifuge tube to which was added poly A+mRNA (7 μl, 5 μg).The reaction tube was heated to 70° C. for 5 minutes or time sufficientto denature the secondary structure of the mRNA. The reaction was thenchilled on ice and 5×First strand buffer (Life Tech., 4 μl), 0.1 M DTT(2 μl) and 10 mM dNTP Mix (Life Tech., 1 μl) were added and then heatedto 37° C. for 2 minutes to equilibrate the temperature. Superscript II®reverse transcriptase (Life Tech., 5 μl) was then added, the reactiontube mixed well and incubated at 37° C. for 1 hour, and terminated byplacement on ice. The final concentration of the reactants was thefollowing: 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl₂; 10 mM DTT;500 μM each dATP, dCTP, dGTP and dTTP; 50 μg/ml Not 1 primer-adapter; 5μg (250 μg/ml) mRNA; 50,000 U/ml Superscript II® reverse transcriptase.

Second Strand Synthesis:

While on ice, the following reagents were added to the reaction tubefrom the first strand synthesis, the reaction well mixed and allowed toreact at 16° C. for 2 hours, taking care not to allow the temperature togo above 160° C.: distilled water (93 μl); 5×Second strand buffer (30μl); dNTP mix (3 μl); 10 U/μl E. Coli DNA ligase (1 μl); 10 U/μl E. ColiDNA polymerase I (4 μl); 2 U/μl E. Coli RNase H (1 μl). 10 U T4 DNAPolymerase (2 μl) was added and the reaction continued to incubate at16° C. for another 5 minutes. The final concentration of the reactionwas the following: 25 mM Tris-HCl (pH 7.5); 100 mM KCl; 5 mM MgCl₂; 10mM (NH₄)₂SO₄; 0.15 mM β-NAD+; 250 μM each dATP, dCTP, dGTP, dTTP; 1.2 mMDTT; 65 U/ml DNA ligase; 250 U/ml DNA polymerase I; 13 U/ml Rnase H. Thereaction has halted by placement on ice and by addition of 0.5 M EDTA(10 μl), then extracted through phenol:chloroform:isoamyl alcohol(25:24:1, 150 μl). The aqueous phase was removed, collected and dilutedinto 5M NaCl (15 μl) and absolute ethanol (−20° C., 400 μl) andcentrifuged for 2 minutes at 14,000 ×g. The supernatant was carefullyremoved from the resulting DNA pellet, the pellet resuspended in 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 ×g. Thesupernatant was again removed and the pellet dried in a speedvac.

Adapter Addition

The following reagents were added to the cDNA pellet from the Secondstrand synthesis above, and the reaction was gently mixed and incubatedat 16° C. for 16 hours: distilled water (25 μl); 5×T4 DNA ligase buffer(10 μl); Sal I adapters (10 μl); T4 DNA ligase (5 μl). The finalcomposition of the reaction was the following: 50 mM Tris-HCl (pH 7.6);10 mM MgCl; 1 mM ATP; 5% (w/v) PEG 8000; 1 mM DTT; 200 μg/ml Sal 1adapters; 100 U/ml T4 DNA ligase. The reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 50 μl), the aqueous phaseremoved, collected and diluted into 5M NaCl (8 μl ) and absolute ethanol(−20° C., 250 μl). This was then centrifuged for 20 minutes at 14,000×g, the supernatant removed and the pellet was resuspended in 0.5 ml 70%ethanol, and centrifuged again for 2 minutes at 14,000×g. Subsequently,the supernatant was removed and the resulting pellet dried in a speedvacand carried on into the next procedure.

Enzymatic Digestion:

To the cDNA prepared with the Sal 1 adapter from the previous paragraphwas added the following reagents and the mixture was incubated at 37° C.for 2 hours: DEPC-treated water (41 μl); Not 1 restriction buffer(REACT, Life Tech., 5 μl), Not 1 (4 μl). The final composition of thisreaction was the following: 50 mM Tris-HCl (pH 8.0); 10 mM MgCl₂; 100 mMMaCl; 1,200 U/ml Not 1.

Gel Isolation of cDNA:

The cDNA is size fractionated by acrylamide gel electrophoresis on a 5%acrylamide gel, and any fragments which were larger than 1 Kb, asdetermined by comparison with a molecular weight marker, were excisedfrom the gel. The cDNA was then electroeluted from the gel into 0.1×TBEbuffer (200 μl) and extracted with phenol:chloroform:isoamyl alcohol(25:24:1, 200 μl). The aqueous phase was removed, collected andcentrifuged for 20 minutes at 14,000 ×g. The supernatant was removedfrom the DNA pellet which was resuspended in 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000 ×g. The supernatant was againdiscarded, the pellet dried in a speedvac and resuspended in distilledwater (15 μl).

Ligation of cDNA into pRK5 Vector:

The following reagents were added together and incubated at 16° C. for16 hours: 5×T4 ligase buffer (3 μl); pRK5, Xho1, Not1 digested vector,0.5 μg, 1 μl); cDNA prepared from previous paragraph (5 μl) anddistilled water (6 μl). Subsequently, additional distilled water (70 μl)and 10 mg/ml tRNA (0.1 μl) were added and the entire reaction wasextracted through phenol;chloroform;isoamyl alcohol (25:24;1). Theaqueous phase was removed, collected and diluted into 5M NaCl (10 μl)and absolute ethanol (−20° C., 250 μl). This was then centrifuged for 20minutes at 14,000 ×g, decanted, and the pellet resuspended into 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 ×g. TheDNA pellet was then dried in a speedvac and eluted into distilled water(3 μl) for use in the subsequent procedure.

Transformation of Library Ligation Into Bacteria:

The ligated cDNA/pRK5 vector DNA prepared previously was chilled on iceto which was added electrocompetent DH10B bacteria (Life Tech., 20 μl).The bacteria vector mixture was then electroporated as per themanufacturers recommendation. Subsequently SOC media (1 ml) was addedand the mixture was incubated at 37° C. for 30 minutes. Thetransformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (370° C.) to allow thecolonies to grow. Positive colonies were then scraped off and the DNAisolated from the bacterial pellet using standard CsCl-gradientprotocols. For example, Ausubel et al., 2.3.1.

Identification of FLS 139

FLS139 can be identified in the human fetal liver library by anystandard method known in the art, including the methods reported byKlein R.D. et al. (1996), Proc. Natl. Acad. Sci. 93, 7108-7113 andJacobs (U.S. Pat. No. 5,563,637 issued Jul. 16, 1996). According toKlien et. al. and Jacobs, cDNAs encoding novel secreted andmembrane-bound mammalian proteins are identified by detecting theirsecretory leader sequences using the yeast invertase gene as a reportersystem. The enzyme invertase catalyzes the breakdown of sucrose toglucose and fructose as well as the breakdown of raffinose to sucroseand melibiose. The secreted form of invertase is required for theutilization of sucrose by yeast (Saccharomyces cerevisiae) so that yeastcells that are unable to produce secreted invertase grow poorly on mediacontaining sucrose as the sole carbon and energy source. Both Klein R.D., supra, and Jacobs, supra, take advantage of the known ability ofmammalian signal sequences to functionally replace the native signalsequence of yeast invertase. A mammalian cDNA library is ligated to aDNA encoding a nonsecreted yeast invertase, the ligated DNA is isolatedand transformed into yeast cells that do not contain an invertase gene.Recombinants containing the nonsecreted yeast invertase gene ligated toa mammalian signal sequence are identified based upon their ability togrow on a medium containing only sucrose or only raffinose as the carbonsource. The mammalian signal sequences identified are then used toscreen a second, full-length cDNA library to isolate the full-lengthclones encoding the corresponding secreted proteins. Cloning may, forexample, be performed by expression cloning or by any other techniqueknown in the art.

The primers used for the identification of FL139 are as follows:

OLI114 CCACGTTGGCTTGAAATTGA SEQ. ID. NO: 13

OLI115 CCTCCAGAATTGATCAAGACAATTCATGATTTGATTCTCTATCTCCAGAG SEQ.ID NO: 14

OLI116 TCGTCTAACATAGCAAATC SEQ. ID. NO:15

The nucleotide sequence of FLS 139 is shown in FIGS. 6A and 6B (SEQ. ID.NO: 5), while its amino acid sequence is shown in FIGS. 7A and 7B (SEQ.ID. NO:6). As illustrated in FIG. 1, FLS 139 contains a fibrinogen-likedomain exhibiting a high degree of sequence homology with the two knownhuman ligands of the 1TIE-2 receptor (h-TIE2L1 and h-TIE2L2).Accordingly, FLS 139 has been identified as a novel member of the TIEligand family.

A clone of FLS139 was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209on Sep. 18, 1997 under the terms of the Budapest Treaty, and has beenassigned the deposit number 209281.

EXAMPLE 2

Identification of NL2 and NL3

NL2 and NL3 were by screening the GenBank database using the computerprogram BLAST (Altshul et al., Methods in Enzymology 266:460480 (1996).The NL2 sequence shows homology with known EST sequences T08223,AA122061, and M62290. Similarly, NL3 shows homology with the known ESTsequences T57280, and T50719. None of the known EST sequences have beenidentified as full length sequences, or described as ligands associatedwith the TIE receptors.

Following their identification, NL2 and NL3 were cloned from a humanfetal lung library prepared from mRNA purchased from Clontech, Inc.(Palo Alto, Calif., USA), catalog # 6528-1, following the manufacturer'sinstructions. The library was screened by hybridization with syntheticoligonucleotide probes:

For NL2:

NL2,5-1 ATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGC SEQ. ID. NO: 7

NL2,3-1 CAACTGGCTGGGCCATCTCGGGCAGCCTCTTTCTTCGGG SEQ. ID. NO: 8

NL2,34 CCCAGCCAGAACTCGCCGTGGGGA SEQ. ID. NO: 9

For NL3:

NL3,5-1 TGGTTGGCAAAGGCAAGGTGGCTGACGATCCGG SEQ. ID. NO: 10

NL3,3-1 GTGGCCCTTATCTCTCCTGTACAGCTTCCGGATCGTCAGCCAC SEQ. ID. NO: 11

NL3,3-2 TCCATTCCCACCTATGACGCTGACCCA SEQ. ID. NO: 12

based on the ESTs found in the GenBank database. cDNA sequences weresequences in their entireties.

The nucleotide and amino acid sequences of NL2 are shown in FIGS. 2A and2B (SEQ. ID. NO: 1) and FIGS. 3A and 3B (SEQ. ID. NO: 2), respectively.The nucleotide and amino acid sequences of NL3 are shown in FIG. 4 (SEQ.ID. NO: 3) and FIG. 5 (SEQ. ID. NO: 4), respectively.

A clone of NL2 (NL2-DNA 22780-1078) was deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20852, on Sep. 18, 1997 under the terms of the Budapest Treaty, and hasbeen assigned the deposit number 209284.

A clone of NL3 was deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Massassas, Va. 20110-2209 on Sep.18, 1997 under the terms of the Budapest Treaty, and has been assignedthe deposit number 209283.

EXAMPLE 3

Northern Blot Analysis

Expression of the FLS139, NL2 and NL3 mRNA in human tissues was examinedby Northern blot analysis. Human mRNA blots were hybridized to a³²P-labeled DNA probe based on the full length cDNAs; the probes weregenerated by digesting and purifying the cDNA inserts. Human fetal RNAblot MTN (Clontech) and human adult RNA blot MTN-II (Clontech) wereincubated with the DNA probes. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2Denhardt's solution; 100 mg/mL denaturedsheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42° C.The blots were washed several times in 2×SSC; 0.05% SDS for 1 hour atroom temperature, followed by a 30 minute wash in 0. 1×SSC; 0.1% SDS at50° C. The blots were developed after overnight exposure byphosphorimager analysis (Fuji).

As shown in FIGS. 8 and 9, NL2 and NL3 mRNA transcripts were detected.

EXAMPLE 4 Expression of FLS 139, NL-2 and NL-3 in E. coli

This example illustrates the preparation of an unglycosylated form ofthe TIE ligands of the present invention in E. coli. The DNA sequenceencoding a NL-2, NL-3 or FLS 139 ligand (SEQ. ID. NOs: 1, 3, and 5,respectively) is initially amplified using selected PCR primers. Theprimers should contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector. A variety ofexpression vectors may be employed. The vector will preferably encode anantibiotic resistance gene, an origin of replication, e promoter, and aribozyme binding site. An example of a suitable vector is pBR322(derived from E. coli; see Bolivar et al., Gene 2:95 (1977)) whichcontains genes for ampicillin and tetracycline resistance. The vector isdigested with restriction enzyme and dephosphorylated. The PCR amplifiedsequences are then ligated into the vector.

The ligation mixture is then used to transform a selected E. colistrain, such as . . . , using the methods described in Sambrook et al.,supra. Transformants are identified by their ability to grow on LBplates and antibiotic resistant colonies are then selected. Plasmid DNAcan be isolated and confirmed by restriction analysis.

Selected clones can be grown overnight in liquid culture medium such as1B broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a later scale culture. The cells arethen grown to a desired optical density. An inducer, such as IPTG may beadded.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized protein can then be purified using a metal chelatingcolumn under conditions that allow tight binding of the protein.

EXAMPLE 5

Expression of FLS 139, NL2 and NL3 in Mammalian Cells

This example illustrates preparation of a glycosylated form of the FLS139, NL2 and NL3 ligands by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the FLS 139, NL2 and NL3 DNA isligated into pRK5 with selected restriction enzymes to allow insertionof the FLS 139, NL2 and NL3 DNA using ligation methods such as describedin Sambrook et al., supra. The resulting vector is called pRK5-FLS 139,-NL2 and NL3, respectively.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-FLS139, -NL2 and NL-3 DNA is mixed with about 1 μg DNA encoding theVA RNA gene [Thimmappaya et al., Cell. 31:543 (1982)] and dissolved in500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture isadded, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mMNaPO₄, and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of FLS139, NL2 and NL3 polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

In an alternative technique, FLS 139, NL2 and NL3 may be introduced into293 cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 pg pRK5-FLS139, -NL2and -NL3 DNA is added. The cells are first concentrated from the spinnerflask by centrifugation and washed with PBS. The DNA-dextran precipitateis incubated on the cell pellet for four hours. The cells are treatedwith 20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μ/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed FLS 139, NL2 and NL3 canthen be concentrated and purified by any selected method, such asdialysis and/or column chromatography.

In another embodiment, FLS 139, NL2 and NL3 can be expressed in CHOcells. The pRK5-FLS 139, -NL2 and -NL3 can be transfected into CHO cellsusing known reagents such as CaPO₄ or DEAE-dextran. As described above,the cell cultures can be incubated, and the medium replaced with culturemedium (alone) or medium containing a radiolabel such as ³⁵S-methionine.After determining the presence of FLS 139, NL2 and NL3 polypeptide, theculture medium may be replaced with serum free medium. Preferably, thecultures are incubated for about 6 days, and then the conditioned mediumis harvested. The medium containing the expressed FLS 139, NL2 and NL3can then be concentrated and purified by any selected method.

Epitope-tagged FLS 139, NL2 and NL3 may also be expressed in host CHOcells. FLS 139, NL2 and NL3 may be subcloned out of the pRK5 vector. Thesubclone insert can undergo PCR to fuse in frame with a selected epitopetag such as a poly-his tag into a Baculovirus expression vector. Thepoly-his tagged FLS139, NL2 and NL3 insert can then be subcloned into aSV40 driven vector containing a selection marker such as DHFR forselection of stable clones. Finally, the CHO cells can be transfected(as described above) with the SV40 driven vector. Labeling may beperformed, as described above, to verify expression. The culture mediumcontaining the expressed poly-His tagged FLS 139, NL2 and NL3 can thenbe concentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

EXAMPLE 6

Expression of FLS 139, NL2 and NL3 in Yeast

First, yeast expression vectors are constructed for intracellularproduction or secretion of FLS 139, NL2 and NL3 from the ADH2/GAPDHpromoter. DNA encoding FLS 139, NL2 and NL3, a selected signal peptideand the promoter is inserted into suitable restriction enzyme sites inthe selected plasmid to direct intracellular expression of FLS 139, NL2and NL3. For secretion, DNA encoding FLS139, NL2 and NL3 can be clonedinto the selected plasmid, together with DNA encoding the ADH2/GAPDHpromoter, the yeast alpha-factor secretory signalAeader sequence, andlinker sequences (if needed) for expression of FLS 139, NL2 and NL3.

Yeast cells, such as yeast strain AB 110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant FLS 139, NL2 and NL3 can subsequently be isolated andpurified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing FLS 139, NL2 and NL3 mayfurther be purified using selected column chromatography resins.

EXAMPLE 7

Expression of FLS 139, NL2 and NL3 in Baculovirus

The following method describes recombinant expression of FLS 139, NL2and NL3 in Baculovirus.

The FLS 139, NL2 and NL3 is fused upstream of an epitope tag containedwith a baculovirus expression vector. Such epitope tags include poly-histags and immunoglobulin tags (like Fc regions of IgG). A variety ofplasmids may be employed, including plasmids derived from commerciallyavailable plasmids such as pVL1393 (Novagen). Briefly, the FLS 139, NL2and NL3 or the desired portion of the FLS 139, NL2 and NL3 (such as thesequence encoding the extracellular domain of a transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged FLS 139, NL2 and NL3 can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged FLS139, NL2 and NL3 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) FLS 139,NL2 and NL3 can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

EXAMPLE 8

Preparation of Antibodies That Bind FLS 139, NL2, or NL3

This example illustrates preparation of monoclonal antibodies which canspecifically bind FLS 139, NL2, or NL3.

Techniques for producing the monoclonal antibodies are known in the artand are described, for example, in Goding, supra. Immunogens that may beemployed include purified ligands of the present invention, fusionproteins containing such ligands, and cells expressing recombinantligands on the cell surface. Selection of the immunogen can be made bythe skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind foodpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice might also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing ELISA assays to detectthe antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of the given ligand. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3×63AgU. 1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, arninopterin, andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe antigen. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against the TIE ligands herein is wellwithin the skill in the art.

The positive hybridoma cells can be injected intraperitoneal intosyngeneic Balb/c mice to produce ascites containing the anti-TIE-ligandmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Deposit of Material

As noted before, the following materials have been deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 USA (ATCC):

Material ATCC Dep,. No. Deposit Date NL2-DNA 22780-1078 209284 9/18/97NL3-DNA 33457-1078 209283 9/18/97 FLS139-DNA 16451-1978 209281 9/18/97

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of the deposit. The deposit will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC §122 and Commissioner's rulespursuant thereto (including 37 C.F.R. §1.14 with particular reference to886 OG 683).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die to be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The present specification is considered to be sufficient to enable oneskilled in the art to practice the invention. The present invention isnot to be limited in scope by the construct deposited, since thedeposited embodiment is intended as a single illustration of certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of the invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription is inadequate to enable the practice of any aspect of theinvention, including the best more thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustrations that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

15 1 1869 DNA Homo sapiens 1 gccgagctga gcggatcctc acatgactgt gatccgattctttccagcgg cttctgcaac 60 caagcgggtc ttacccccgg tcctccgcgt ctccagtcctcgcacctgga accccaacgt 120 ccccgagagt ccccgaatcc ccgctcccag gctacctaagaggatgagcg gtgctccgac 180 ggccggggca gccctgatgc tctgcgccgc caccgccgtgctactgagcg ctcagggcgg 240 acccgtgcag tccaagtcgc cgcgctttgc gtcctgggacgagatgaatg tcctggcgca 300 cggactcctg cagctcggcc aggggctgcg cgaacacgcggagcgcaccc gcagtcagct 360 gagcgcgctg gagcggcgcc tgagcgcgtg cgggtccgcctgtcagggaa ccgaggggtc 420 caccgacctc ccgttagccc ctgagagccg ggtggaccctgaggtccttc acagcctgca 480 gacacaactc aaggctcaga acagcaggat ccagcaactcttccacaagg tggcccagca 540 gcagcggcac ctggagaagc agcacctgcg aattcagcatctgcaaagcc agtttggcct 600 cctggaccac aagcacctag accatgaggt ggccaagcctgcccgaagaa agaggctgcc 660 cgagatggcc cagccagttg acccggctca caatgtcagccgcctgcacc ggctgcccag 720 ggattgccag gagctgttcc aggttgggga gaggcagagtggactatttg aaatccagcc 780 tcaggggtct ccgccatttt tggtgaactg caagatgacctcagatggag gctggacagt 840 aattcagagg cgccacgatg gctcagtgga cttcaaccggccctgggaag cctacaaggc 900 ggggtttggg gatccccacg gcgagttctg gctgggtctggagaaggtgc atagcatcac 960 gggggaccgc aacagccgcc tggccgtgca gctgcgggactgggatggca acgccgagtt 1020 gctgcagttc tccgtgcacc tgggtggcga ggacacggcctatagcctgc agctcactgc 1080 acccgtggcc ggccagctgg gcgccaccac cgtcccacccagcggcctct ccgtaccctt 1140 ctccacttgg gaccaggatc acgacctccg cagggacaagaactgcgcca agagcctctc 1200 tggaggctgg tggtttggca cctgcagcca ttccaacctcaacggccagt acttccgctc 1260 catcccacag cagcggcaga agcttaagaa gggaatcttctggaagacct ggcggggccg 1320 ctactacccg ctgcaggcca ccaccatgtt gatccagcccatggcagcag aggcagcctc 1380 ctagcgtcct ggctgggcct ggtcccaggc ccacgaaagacggtgactct tggctctgcc 1440 cgaggatgtg gccgttccct gcctgggcag gggctccaaggaggggccat ctggaaactt 1500 gtggacagag aagaagacca cgactggaga agccccctttctgagtgcag gggggctgca 1560 tgcgttgcct cctgagatcg aggctgcagg atatgctcagactctagagg cgtggaccaa 1620 ggggcatgga gcttcactcc ttgctggcca gggagttggggactcagagg gaccacttgg 1680 ggccagccag actggcctca atggcggact cagtcacattgactgacggg gaccagggct 1740 tgtgtgggtc gagagcgccc tcatggtgct ggtgctgttgtgtgtaggtc ccctggggac 1800 acaagcaggc gccaatggta tctgggcgga gctcacagagttcttggaat aaaagcaacc 1860 tcagaacac 1869 2 406 PRT Homo sapiens UNSURE(0)...(0) Xaa = any amino acid 2 Met Ser Gly Ala Pro Thr Ala Gly Ala AlaLeu Met Leu Cys Ala Ala 1 5 10 15 Thr Ala Val Leu Leu Ser Ala Gln GlyGly Pro Val Gln Ser Lys Ser 20 25 30 Pro Arg Phe Ala Ser Trp Asp Glu MetAsn Val Leu Ala His Gly Leu 35 40 45 Leu Gln Leu Gly Gln Gly Leu Arg GluHis Ala Glu Arg Thr Arg Ser 50 55 60 Gln Leu Ser Ala Leu Glu Arg Arg LeuSer Ala Cys Gly Ser Ala Cys 65 70 75 80 Gln Gly Thr Glu Gly Ser Thr AspLeu Pro Leu Ala Pro Glu Ser Arg 85 90 95 Val Asp Pro Glu Val Leu His SerLeu Gln Thr Gln Leu Lys Ala Gln 100 105 110 Asn Ser Arg Ile Gln Gln LeuPhe His Lys Val Ala Gln Gln Gln Arg 115 120 125 His Leu Glu Lys Gln HisLeu Arg Ile Gln His Leu Gln Ser Gln Phe 130 135 140 Gly Leu Leu Asp HisLys His Leu Asp His Glu Val Ala Lys Pro Ala 145 150 155 160 Arg Arg LysArg Leu Pro Glu Met Ala Gln Pro Val Asp Pro Ala His 165 170 175 Asn ValSer Arg Leu His Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe 180 185 190 GlnVal Gly Glu Arg Gln Ser Gly Leu Phe Glu Ile Gln Pro Gln Gly 195 200 205Ser Pro Pro Phe Leu Val Asn Cys Lys Met Thr Ser Xaa Gly Gly Trp 210 215220 Thr Val Ile Gln Arg Arg His Asp Gly Ser Val Asp Phe Asn Arg Pro 225230 235 240 Trp Glu Ala Tyr Lys Ala Gly Phe Gly Asp Pro His Gly Glu PheTrp 245 250 255 Leu Gly Leu Glu Lys Val His Ser Ile Thr Gly Asp Arg AsnSer Arg 260 265 270 Leu Ala Val Gln Leu Arg Asp Trp Asp Gly Asn Ala GluLeu Leu Gln 275 280 285 Phe Ser Val His Leu Gly Gly Glu Asp Thr Ala TyrSer Leu Gln Leu 290 295 300 Thr Ala Pro Val Ala Gly Gln Leu Gly Ala ThrThr Val Pro Pro Ser 305 310 315 320 Gly Leu Ser Val Pro Phe Ser Thr TrpAsp Gln Asp His Asn Leu Arg 325 330 335 Arg Asp Lys Asn Cys Ala Lys SerLeu Ser Gly Gly Trp Trp Phe Gly 340 345 350 Thr Cys Ser His Ser Asn LeuAsn Gly Gln Tyr Phe Arg Ser Ile Pro 355 360 365 Gln Gln Arg Gln Lys LeuLys Lys Gly Ile Phe Trp Lys Thr Trp Arg 370 375 380 Gly Arg Tyr Tyr ProLeu Gln Ala Thr Thr Met Leu Ile Gln Pro Met 385 390 395 400 Ala Ala GluAla Ala Ser 405 3 1024 DNA Homo sapiens 3 cggacgcgtg ggcccctggtgggcccagca agatggatct actgtggatc ctgccctccc 60 tgtggcttct cctgcttggggggcctgcct gcctgaagac ccaggaacac cccagctgcc 120 caggacccag ggaactggaagccagcaaag ttgtcctcct gcccagttgt cccggagctc 180 caggaagtcc tggggagaagggagccccag gtcctcaagg gccacctgga ccaccaggca 240 agatgggccc caagggtgagccaggcccca gaaactgccg ggagctgttg agccagggcg 300 ccaccttgag cggctggtaccatctgtgcc tacctgaggg cagggccctc ccagtctttt 360 gtgacatgga caccgaggggggcggctggc tggtgtttca gaggcgccag gatggttctg 420 tggatttctt ccgctcttggtcctcctaca gagcaggttt tgggaaccaa gagtctgaat 480 tctggctggg aaatgagaatttgcaccagc ttactctcca gggtaactgg gagctgcggg 540 tagagctgga agactttaatggtaaccgta ctttcgccca ctatgcgacc ttccgcctcc 600 tcggtgaggt agaccactaccagctggcac tgggcaagtt ctcagagggc actgcagggg 660 attccctgag cctccacagtgggaggccct ttaccaccta tgacgctgac cacgattcaa 720 gcaacagcaa ctgtgcagtgattgtccacg gtgcctggtg gtatgcatcc tgttaccgat 780 caaatctcaa tggtcgctatgcagtgtctg aggctgccgc ccacaaatat ggcattgact 840 gggcctcagg ccgtggtgtgggccacccct accgcagggt tcggatgatg cttcgatagg 900 gcactctggc agccagtgcccttatctctc ctgtacagct tccggatcgt cagccacctt 960 gcctttgcca accacctctgcttgcctgtc cacatttaaa aataaaatca ttttagccct 1020 ttca 1024 4 288 PRTHomo sapiens 4 Met Asp Leu Leu Trp Ile Leu Pro Ser Leu Trp Leu Leu LeuLeu Gly 1 5 10 15 Gly Pro Ala Cys Leu Lys Thr Gln Glu His Pro Ser CysPro Gly Pro 20 25 30 Arg Glu Leu Glu Ala Ser Lys Val Val Leu Leu Pro SerCys Pro Gly 35 40 45 Ala Pro Gly Ser Pro Gly Glu Lys Gly Ala Pro Gly ProGln Gly Pro 50 55 60 Pro Gly Pro Pro Gly Lys Met Gly Pro Lys Gly Glu ProGly Pro Arg 65 70 75 80 Asn Cys Arg Glu Leu Leu Ser Gln Gly Ala Thr LeuSer Gly Trp Tyr 85 90 95 His Leu Cys Leu Pro Glu Gly Arg Ala Leu Pro ValPhe Cys Asp Met 100 105 110 Asp Thr Glu Gly Gly Gly Trp Leu Val Phe GlnArg Arg Gln Asp Gly 115 120 125 Ser Val Asp Phe Phe Arg Ser Trp Ser SerTyr Arg Ala Gly Phe Gly 130 135 140 Asn Gln Glu Ser Glu Phe Trp Leu GlyAsn Glu Asn Leu His Gln Leu 145 150 155 160 Thr Leu Gln Gly Asn Trp GluLeu Arg Val Glu Leu Glu Asp Phe Asn 165 170 175 Gly Asn Arg Thr Phe AlaHis Tyr Ala Thr Phe Arg Leu Leu Gly Glu 180 185 190 Val Asp His Tyr GlnLeu Ala Leu Gly Lys Phe Ser Glu Gly Thr Ala 195 200 205 Gly Asp Ser LeuSer Leu His Ser Gly Arg Pro Phe Thr Thr Tyr Asp 210 215 220 Ala Asp HisAsp Ser Ser Asn Ser Asn Cys Ala Val Ile Val His Gly 225 230 235 240 AlaTrp Trp Tyr Ala Ser Cys Tyr Arg Ser Asn Leu Asn Gly Arg Tyr 245 250 255Ala Val Ser Glu Ala Ala Ala His Lys Tyr Gly Ile Asp Trp Ala Ser 260 265270 Gly Arg Gly Val Gly His Pro Tyr Arg Arg Val Arg Met Met Leu Arg 275280 285 5 2042 DNA Homo sapiens 5 gcggacgcgt gggtgaaatt gaaaatcaagataaaaatgt tcacaattaa gctccttctt 60 tttattgttc ctctagttat ttcctccagaattgatcaag acaattcatc atttgattct 120 ctatctccag agccaaaatc aagatttgctatgttagacg atgtaaaaat tttagccaat 180 ggcctccttc agttgggaca tggtcttaaagactttgtcc ataagacgaa gggccaaatt 240 aatgacatat ttcaaaaact caacatatttgatcagtctt tttatgatct atcgctgcaa 300 accagtgaaa tcaaagaaga agaaaaggaactgagaagaa ctacatataa actacaagtc 360 aaaaatgaag aggtaaagaa tatgtcacttgaactcaact caaaacttga aagcctccta 420 gaagaaaaaa ttctacttca acaaaaagtgaaatatttag aagagcaact aactaactta 480 attcaaaatc aacctgaaac tccagaacacccagaagtaa cttcacttaa aacttttgta 540 gaaaaacaag ataatagcat caaagaccttctccagaccg tggaagacca atataaacaa 600 ttaaaccaac agcatagtca aataaaagaaatagaaaatc agctcagaag gactagtatt 660 caagaaccca cagaaatttc tctatcttccaagccaagag caccaagaac tactcccttt 720 cttcagttga atgaaataag aaatgtaaaacatgatggca ttcctgctga atgtaccacc 780 atttataaca gaggtgaaca tacaagtggcatgtatgcca tcagacccag caactctcaa 840 gtttttcatg tctactgtga tgttatatcaggtagtccat ggacattaat tcaacatcga 900 atagatggat cacaaaactt caatgaaacgtgggagaact acaaatatgg ttttgggagg 960 cttgatggag aattttggtt gggcctagagaagatatact ccatagtgaa gcaatctaat 1020 tatgttttac gaattgagtt ggaagactggaaagacaaca aacattatat tgaatattct 1080 ttttacttgg gaaatcacga aaccaactatacgctacatc tagttgcgat tactggcaat 1140 gtccccaatg caatcccgga aaacaaagatttggtgtttt ctacttggga tcacaaagca 1200 aaaggacact tcaactgtcc agagggttattcaggaggct ggtggtggca tgatgagtgt 1260 ggagaaaaca acctaaatgg taaatataacaaaccaagag caaaatctaa gccagagagg 1320 agaagaggat tatcttggaa gtctcaaaatggaaggttat actctataaa atcaaccaaa 1380 atgttgatcc atccaacaga ttcagaaagctttgaatgaa ctgaggcaat ttaaaggcat 1440 atttaaccat taactcattc caagttaatgtggtctaata atctggtata aatccttaag 1500 agaaagcttg agaaatagat tttttttatcttaaagtcac tgtctattta agattaaaca 1560 tacaatcaca taaccttaaa gaataccgtttacatttctc aatcaaaatt cttataatac 1620 tatttgtttt aaattttgtg atgtgggaatcaattttaga tggtcacaat ctagattata 1680 atcaataggt gaacttatta aataacttttctaaataaaa aatttagaga cttttatttt 1740 aaaaggcatc atatgagcta atatcacaactttcccagtt taaaaaacta gtactcttgt 1800 taaaactcta aacttgacta aatacagaggactggtaatt gtacagttct taaatgttgt 1860 agtattaatt tcaaaactaa aaatcgtcagcacagagtat gtgtaaaaat ctgtaataca 1920 aatttttaaa ctgatgcttc attttgctacaaaataattt ggagtaaatg tttgatatga 1980 tttatttatg aaacctaatg aagcagaattaaatactgta ttaaaataag ttcgctgtct 2040 tt 2042 6 460 PRT Homo sapiens 6Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 1015 Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 2530 Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 4045 Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 5560 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 7075 80 Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu 8590 95 Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu100 105 110 Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser LeuLeu 115 120 125 Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu GluGlu Gln 130 135 140 Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro GluHis Pro Glu 145 150 155 160 Val Thr Ser Leu Lys Thr Phe Val Glu Lys GlnAsp Asn Ser Ile Lys 165 170 175 Asp Leu Leu Gln Thr Val Glu Asp Gln TyrLys Gln Leu Asn Gln Gln 180 185 190 His Ser Gln Ile Lys Glu Ile Glu AsnGln Leu Arg Arg Thr Ser Ile 195 200 205 Gln Glu Pro Thr Glu Ile Ser LeuSer Ser Lys Pro Arg Ala Pro Arg 210 215 220 Thr Thr Pro Phe Leu Gln LeuAsn Glu Ile Arg Asn Val Lys His Asp 225 230 235 240 Gly Ile Pro Ala GluCys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Met TyrAla Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 260 265 270 Tyr Cys AspVal Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 Ile AspGly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300 GlyPhe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 305 310 315320 Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 325330 335 Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly340 345 350 Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr GlyAsn 355 360 365 Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe SerThr Trp 370 375 380 Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu GlyTyr Ser Gly 385 390 395 400 Gly Trp Trp Trp His Asp Glu Cys Gly Glu AsnAsn Leu Asn Gly Lys 405 410 415 Tyr Asn Lys Pro Arg Ala Lys Ser Lys ProGlu Arg Arg Arg Gly Leu 420 425 430 Ser Trp Lys Ser Gln Asn Gly Arg LeuTyr Ser Ile Lys Ser Thr Lys 435 440 445 Met Leu Ile His Pro Thr Asp SerGlu Ser Phe Glu 450 455 460 7 33 DNA Homo sapiens 7 atgaggtggccaagcctgcc cgaagaaaga ggc 33 8 39 DNA Homo sapiens 8 caactggctgggccatctcg ggcagcctct ttcttcggg 39 9 24 DNA Homo sapiens 9 cccagccagaactcgccgtg ggga 24 10 33 DNA Homo sapiens 10 tggttggcaa aggcaaggtggctgacgatc cgg 33 11 43 DNA Homo sapiens 11 gtggccctta tctctcctgtacagcttccg gatcgtcagc cac 43 12 27 DNA Homo sapiens 12 tccattcccacctatgacgc tgaccca 27 13 20 DNA Homo sapiens 13 ccacgttggc ttgaaattga 2014 50 DNA Homo sapiens 14 cctccagaat tgatcaagac aattcatgat ttgattctctatctccagag 50 15 19 DNA Homo sapiens 15 tcgtctaaca tagcaaatc 19

What is claimed is:
 1. An isolated nucleic acid molecule which encodes apolypeptide comprising an amino acid sequence having at least 90%sequence identity to the native human NL3 amino acid sequence shown inFIG. 5 (SEQ ID NO: 4), said polypeptide having the ability to inducevascularization.
 2. The isolated nucleic acid molecule of claim 1,wherein said polypeptide comprises an amino acid sequence having atleast 95% sequence identity to the native human NL3 amino acid sequenceshown in FIG. 5 (SEQ ID NO: 4).
 3. The isolated nucleic acid molecule ofclaim 1, wherein said polypeptide comprises an amino acid sequencehaving at least 98% sequence identity to the native human NL3 amino acidsequence shown in FIG. 5 (SEQ ID NO: 4).
 4. The isolated nucleic acidmolecule of claim 1, wherein said polypeptide comprises an amino acidsequence having at least 99% sequence identity to the native human NL3amino acid sequence shown in FIG. 5 (SEQ ID NO: 4).
 5. The isolatednucleic acid molecule of claim 1, wherein said polypeptide comprises theamino acid sequence shown in FIG. 5 (SEQ ID NO: 4).
 6. The isolatednucleic acid molecule of claim 1, wherein said polypeptide consists ofthe amino acid sequence shown in FIG. 5 (SEQ ID NO: 4).
 7. An isolatednucleic acid molecule which comprises nucleotides 36 to 896 of thenucleotide sequence shown in FIG. 4 (SEQ ID NO: 3).
 8. An isolatednucleic acid molecule which comprises the full-length coding sequence ofthe DNA deposited with the ATCC under accession number
 209283. 9. Anisolated nucleic acid molecule which comprises the coding sequence ofthe fibrinogen-like domain of native human NL3 polypeptide shown in FIG.5 (SEQ ID NO:4).
 10. A vector which comprises the nucleic acid moleculeof any one of claim 1, 7, 8 or
 9. 11. A recombinant host celltransformed with the nucleic acid molecule of claim
 1. 12. Therecombinant host cell of claim 11 which is a prokaryotic cell.
 13. Therecombinant host cell of claim 11 which is a eukaryotic cell.